PAN-DR binding polypeptides and uses thereof

ABSTRACT

The present invention provides novel artificial oligopeptides capable of binding HLA Class II molecules encoded by several alleles. The oligopeptides include the sequence AX 1 FVAAX 2 TLX 3 AX 4 A (SEQ ID NO:1), wherein X 1  is H; X 2  is selected from the group consisting of F, N, Y and W; X 3  is H and X 4  is selected from the group consisting of A, D and E. The invention also relates to large peptides comprising the oligopeptides, polynucleotides encoding the oligopeptides and larger peptides, as well as to compositions comprising the oligopeptides, peptides or polynucleotides. Also disclosed are methods for inducing immune responses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/146,607, filed Jul. 27, 2011, now U.S. Pat. No. 9,249,187, whichis a U.S. National Stage of International Application No.PCT/EP2010/050839, filed Jan. 26, 2010, said International ApplicationNo. PCT/EP2010/050839 claims the benefits of U.S. provisionalapplication No. 61/147,892, and EP application number 09151495.0, bothfiled on Jan. 28, 2009, all of which are herein incorporated byreference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2473_0660002_SeqListingUPDATED_02152017_ST25.txt, Size: 45,883 bytes;and Date of Creation: Feb. 15, 2017) filed herewith was originally filedwith the U.S. application Ser. No. 13/146,607 and is incorporated hereinby reference in its entirety.

BACKGROUND OF THE INVENTION

Immunogenic peptides, containing epitopes recognized by T helper cells,have been found to be useful in inducing immune responses. The use ofhelper peptides to enhance antibody responses against particulardeterminants is described for instance in Hervas-Stubbs, et al., Vaccine12:867-871 (1994).

Although allele-specific polymorphic residues that line the peptidebinding pockets of MHC alleles tend to endow each allele with thecapacity to bind a unique set of peptides, there are instances in whicha given peptide has been shown to bind to more than one MHC allele. Forexample, several investigators reported degenerate binding and/orrecognition of certain epitopes in the context of multiple DR types,leading to the concept that certain peptides might represent “universal”epitopes (Busch, et al., Int. Immunol. 2:443-451 (1990);Panina-Bordignon, et al., Eur. J. Immunol. 19:2237-2242 (1989);Sinigaglia, et al., Nature 336:778-780 (1988); O'Sullivan, et al., J.Immunol. 147:2663-2669 (1991); Roache, et al., J. Immunol. 144:1849-1856(1991); Hill, et al., J. Immunol. 147:189-197 (1991)). Pan-DR bindingpeptides have been described in, for example, U.S. Pat. No. 6,413,935;WO 95/07707; WO/2005/120563; and Alexander, et al., Immunity 1:751-761(1994). These peptides have been shown to help in the generation ofvarious immune responses against antigens.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on studies of the effects of selectedmutations in the sequence of PADRE (SEQ ID NO: 3) in terms of providingimmunogenic peptides (both in the form of short peptides, oligopeptidesand polypeptides) having improved stability vs. proteolytic enzymes.

The invention thus relates to an isolated polypeptide comprising orconsisting of an oligopeptide sequence that can bind an MEW class IImolecule encoded by at least three different HLA-DR alleles with an IC₅₀value of less than 100 nM, such as at least 50 nM, wherein theoligopeptide sequence comprises AX₁FVAAX₂TLX₃AX₄A (SEQ ID NO:1), wherein

X₁ is selected from the group consisting of W, F, Y, H, D, E, N, Q, Iand K;

X₂ is selected from the group consisting of F, N, Y and W;

X₃ is selected from the group consisting of H and K, and

X₄ is selected from the group consisting of A, D and E,

with the proviso that the oligopeptide sequence is not AKFVAAWTLKAAA(SEQ ID NO: 3).

The invention further relates to polynucleotides encoding thepolypeptides of the invention and to vectors including suchpolynucleotides. Also encompassed by the invention are cells comprisingthe polynucleotides of the invention. The invention further relates tocompositions, including pharmaceutical compositions, comprising thepolypeptides or oligopeptides or polynucleotides or vectors or cells ofthe invention. Finally, the invention relates to a method of stimulatingan immune response by administering a polypeptide or oligopeptide orpolynucleotide or vector or cell or composition of the invention to asubject in need thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows data from binding experiments determining the HLA-DRbinding of peptides of the invention to various HLA-DR molecules, cf.Example 1 for details.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Thisinvention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

The terms “oligopeptide” or “peptide” as used herein refer to a chain ofat least four amino acid residues or amino acid mimetics. Theoligopeptides or peptides can be a variety of lengths, either in theirneutral (uncharged) forms or in salt forms, and either free ofmodifications, including but not limited to, glycosylation, side chainoxidation, or phosphorylation or containing one or more of thesemodifications. While optional modifications do not destroy thebiological activity of the polypeptides described herein, however, theinvention includes options in which the modifications reduce oreliminate biological activity (e.g., to limit activity until suchmodification if removed, e.g., in vivo).

The terms “polypeptide,” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. As used herein, the terms encompass aminoacid chains of any length, including full-length proteins (i.e.,antigens), wherein the amino acid residues are linked by covalentpeptide bonds. The terms further include polypeptides which haveundergone post-translational modifications, for example, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. Polypeptides, and fragments, derivatives, analogs,or variants thereof can be antigenic and immunogenic polypeptides.

The terms “amino acid residue,” “amino acid” and “residue” whenreferring to an amino acid residue in a peptide, oligopeptide or proteinare used interchangeably and, as used herein, mean an amino acid oramino acid mimetic joined covalently to at least one other amino acid oramino acid mimetic through an amide bond or amide bond mimetic.

As used herein, the term “amino acid,” when unqualified, refers to an“L-amino acid” or L-amino acid mimetic.

As used herein, the nomenclature used to describe peptide compoundsfollows the conventional practice wherein the amino group is presentedto the left (the N-terminus) and the carboxyl group to the right (theC-terminus) of each amino acid residue. When amino acid residuepositions are referred to in an epitope, they are numbered in an aminoto carboxyl direction with position one being the position closest tothe amino terminal end of the epitope, or the peptide or protein ofwhich it may be a part. In the amino acid structure formulae, eachresidue is generally represented by standard three-letter orsingle-letter designations. The L-form of an amino acid residue isrepresented by a capital single letter or a capital first letter of athree-letter symbol, and the D-form for those amino acids having D-formsis represented by a lower case single letter or a lower case threeletter symbol. Glycine has no asymmetric carbon atom and is simplyreferred to as “Gly” or G.

As used herein, the symbols for the amino acids are shown below.

Single Letter Symbol Three Letter Symbol Amino Acids A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr Tyrosine

The term “pan DR-binding peptide” or “pan-DR binding epitope” as usedherein refers to a member of a family of molecules that binds more thanone MHC class II DR molecule (e.g., binding each of the more than oneMHC molecule with an IC₅₀ of less than 100 nM, such as at least 50 nM).In some embodiments, the pan DR-binding oligopeptides of the presentinvention are peptides capable of binding at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or all 12 most common DR alleles (DR1, 2w2b, 2w2a, 3, 4w4,4w14, 5, 7, 52a, 52b, 52c, and 53). The pan-DR binding oligopeptides ofthe present invention, in addition to promoting an immune responseagainst a second determinant, can also serve as target immunogensthemselves. Thus, for instance, in the case in which the pan-DR bindingpeptide itself is linked to a carbohydrate epitope, the immune responsemay be to both the pan-DR binding peptide and the carbohydrate epitope.

As used herein, the term “PADRE” refers to the pan DR binding peptidehaving the amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 3).

As used herein, the expression “PADRE analogue” refers to a pan DRpeptide comprising an amino acid sequence which, compared to PADRE,includes at most 1, 2, 3 or 4 amino acid changes, of which at least oneis made in K2, W7, K10 or A12.

As used herein, the term “IC₅₀” refers to the concentration of peptidein a binding assay at which 50% inhibition of binding of a referencepeptide is observed. Depending on the conditions in which the assays arerun (i.e., limiting MHC proteins and labeled peptide concentrations),these values may approximate K_(D) values. Assays for determiningbinding are described in detail, e.g., in PCT publications WO 94/20127and WO 94/03205, the disclosure of each which is herein incorporated byreference. It should be noted that IC₅₀ values can change, oftendramatically, if the assay conditions are varied, and depending on theparticular reagents used (e.g., MHC preparation, etc.). For example,excessive concentrations of MHC molecules will increase the apparentmeasured IC₅₀ of a given ligand.

Alternatively, binding is expressed relative to a reference peptide, forinstance PADRE. Although as a particular assay becomes more, or less,sensitive, the IC₅₀ value of the peptides tested may change somewhat,the binding relative to the reference peptide will not significantlychange. For example, in an assay run under conditions such that the IC₅₀of the reference peptide increases 10-fold, the IC₅₀ values of the testpeptides will also shift approximately 10-fold. Therefore, to avoidambiguities, the assessment of whether a peptide is a good,intermediate, weak, or negative binder is generally based on its IC₅₀,relative to the IC₅₀ of a standard peptide. Binding may also bedetermined using other assay systems including those using: live cells(e.g., Ceppellini et al., Nature 339:392, 1989; Christnick et al.,Nature 352:67, 1991; Busch et al., Int. Immunol. 2:443, 19990; Hill etal., J. Immunol. 147:189, 1991; del Guercio et al., J. Immunol. 154:685,1995), cell free systems using detergent lysates (e.g., Cerundolo etal., J. Immunol. 21:2069, 1991), immobilized purified MHC (e.g., Hill etal., J. Immunol. 152, 2890, 1994; Marshall et al., J. Immunol. 152:4946,1994), ELISA systems (e.g., Reay et al., EMBO J. 11:2829, 1992), surfaceplasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425, 1993);high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353,1994), and measurement of class I MHC stabilization or assembly (e.g.,Ljunggren et al., Nature 346:476, 1990; Schumacher et al., Cell 62:563,1990; Townsend et al., Cell 62:285, 1990; Parker et al., J. Immunol.149:1896, 1992).

The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is then translated to form a polypeptide product which hasa relevant biological activity. Also, the process of expression mayinvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

The term “construct” as used herein generally denotes a composition thatdoes not occur in nature. A construct can be produced by synthetictechnologies, e.g., recombinant DNA preparation and expression orchemical synthetic techniques for nucleic or amino acids. A constructcan also be produced by the addition or affiliation of one material withanother such that the result is not found in nature in that form. A“multi-epitope construct” can be used interchangeably with the term“minigene” or “multi-epitope nucleic acid vaccine,” and comprisesmultiple epitope nucleic acids that encode peptide epitopes of anylength that can bind to a molecule functioning in the immune system,e.g., in some embodiments, a MHC class I and a T-cell receptor and/or aMHC class II and a T-cell receptor. Epitope nucleic acids in amulti-epitope construct can encode, for example, class II MHC epitopesor a combination of class I MHC epitopes and class II MHC epitopes.

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vitro or in vivo, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or MHC molecule. Thus, the term “epitope” includes, but isnot limited to, immunogenic peptides of the invention capable of bindingto an appropriate MHC molecule and thereafter inducing a cytotoxic Tcell response, or a helper T cell response, or alternatively, capable ofbinding an antibody, and thereafter inducing an antibody response to theantigen from which the immunogenic peptide is derived.

A “flanking residue” is a residue that is positioned next to an epitope.A flanking residue can be introduced or inserted at a position adjacentto the N-terminus or the C-terminus of an epitope.

The terms “immunogen” and “antigen” are used interchangeably and meanany compound to which a cellular or humoral immune response is to bedirected against. Furthermore, antigenic or immunogenic peptides of theinvention may be linear, i.e., be comprised of contiguous amino acids ina polypeptide, or, in the case of antibody-epitopes, may be threedimensional or conformational, i.e., where a functional epitope iscomprised of non-contiguous amino acids which come together due to thesecondary or tertiary structure of the polypeptide, thereby forming anepitope.

As used herein, the term “antigenic determinant” is any structure thatcan elicit, facilitate, or be induced to produce an immune response, forexample carbohydrate epitopes, lipids, proteins, peptides, orcombinations thereof.

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or genes, that is capable of interacting with andbinding to a specified protein or antigen contained in a compositioncomprising, but not limited to, one or more proteins and/or antigens.

The term “CTL epitope” refers to a peptide, which is recognized andbound by a particular MHC class I molecule, and which is recognized by aT lymphocyte when complexed with the particular MHC Class I molecule. Insome embodiments, the CTL epitope can be from about 8 to about 13 aminoacids in length, from about 9 to about 11 amino acids in length, or fromabout 9 to about 10 amino acids in length.

The term “HTL epitope” refers to a peptide, which is recognized andbound by a particular MHC class II molecule, and which is recognized bya T lymphocyte when complexed with the particular MHC Class II molecule.In some embodiments, the HTL epitope can be from about 6 to about 30amino acids in length, from about 8 to about 30 amino acids in length,from about 10 to about 30 amino acids, from about 12 to about 30 aminoacids in length, from about 6 to about 25 amino acids in length, fromabout 8 to about 25 amino acids in length, from about 10 to about 25amino acids, from about 12 to about 25 amino acids in length, from about6 to about 18 amino acids in length, from about 8 to about 18 aminoacids in length, from about 10 to about 18 amino acids, or from about 12to about 18 amino acids in length.

When stating that a T lymphocyte “recognizes” a complex between an MHCmolecule and a CTL or HTL epitope is herein mean that the T-cellreceptors on the T lymphocyte bind to the complex with the effect thatthe T lymphocyte is activated.

The terms “identical” or percent “identity,” in the context of two ormore peptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesthat are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using a sequence comparisonalgorithm or by manual alignment and visual inspection.

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state.

The term “linker” as used herein is any compound used to providecovalent linkage and spacing between two functional groups (e.g., apan-DR binding peptide and a desired immunogen). In some embodiments,the linker comprises neutral molecules, such as aliphatic carbon chains,amino acids or amino acid mimetics, which are substantially unchargedunder physiological conditions and may have linear or branched sidechains. In some cases, the linker can, itself, be immunogenic. Variouslinkers useful in the invention are described in more detail, below.Additionally, the verbs “link” and “conjugate” are used interchangeablyherein and refer to covalent attachment of two or more species.

The terms “link” or “join” refers to any method known in the art forfunctionally connecting peptides, including, without limitation,recombinant fusion, covalent bonding, disulfide bonding, ionic bonding,hydrogen bonding, and electrostatic bonding.

The term “directly linked” or “directly joined” refers to being joinedwithout anything intervening. For example, in the case of two peptidesbeing directly joined, one peptide would be joined or bonded to anotherpeptide, as described above, without any sequence, molecule, spacer,linker, etc. intervening between the two peptides. Directly joinedpeptides may or may not share amino acids in common, i.e., the sequencesof the two peptides are allowed to not overlap and also to overlap; thelatter is e.g. relevant if a PADRE analogue of the invention isintroduced in another (poly)peptide by means of insertion—in order toensure the presence of the PADRE analogue's amino acid sequence, it isin only necessary to insert those amino acid residues which togetherwith the amino acid residues at the insertion point provides for theentire sequence for the PADRE analogue.

The term “indirectly linked” refers to being joined with somethingintervening. For example, in the case of two peptides being indirectlyjoined, one peptide would be joined or bonded to another peptide, asdescribed above, with a sequence, molecule, spacer, linker, etc.intervening between the two peptides.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, e.g., Paul, Fundamental Immunology, 3rd ed., RavenPress, New York, 1993.

The phrase “pharmaceutically acceptable” refers to a generallynon-toxic, inert, and/or physiologically compatible composition.

DETAILED DESCRIPTION I. Introduction

The inventors have discovered a new class of peptides that are pan-DRbinders, i.e., the peptides bind to MHC class II molecules encoded by anumber of different DR alleles, thereby allowing the peptides tostimulate an immune response in a wide spectrum of individuals. Further,the inventors have discovered that a number of the new peptides havesignificantly improved protease resistance, thereby allowing for alonger in vivo half-life. Surprisingly, some of these peptides reflect agreatly increased ability to boost an immune response over known pan-DRpeptides.

A biological activity of the polypeptides of the invention (or fragmentsthereof) is the ability to bind an appropriate MHC molecule and induce aT helper response, which optionally helps to induce an immune responseagainst a target immunogen or immunogen mimetic. In the case of peptidesuseful for stimulating antibody responses, the peptides of the inventionwill induce a T helper response, which in turn helps induce a humoralresponse against the target immunogen.

II. Pan-DR Binding Peptides

The present invention provides a new class of peptides that are pan-DRbinding peptides and provides uses thereof. The peptides of the presentinvention are capable of binding more than one of a number of differentDR alleles and thus find use in increasing immune responses to variousantigens/immunogens. The peptides of the present invention further finduse in eliciting an enhanced immune response over known pan-DR bindingpeptides, for example, as disclosed in U.S. Pat. No. 5,736,142.

As demonstrated herein, the inventors have identified a series ofpeptide sequences that bind to MHC molecules encoded by multiple HLAalleles. Partly in view of this data, the inventors have determined thatthe sequence motifs below represent pan-DR binding peptides withimproved activity and immunogenicity. In some embodiments, thepolypeptides of the present invention comprise an oligopeptide sequencethat can bind an MHC molecule encoded by at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more different DR alleles, wherein the oligopeptidesequence comprises AX₁FVAAX₂TLX₃AX₄A (SEQ ID NO:1), wherein X₁ isselected from the group consisting of W, F, Y, H, D, E, N, Q, I and K;X₂ is selected from the group consisting of F, N, Y and W; X₃ isselected from the group consisting of H and K, and X₄ is selected fromthe group consisting of A, D and E. In some embodiments, the polypeptidecomprises an oligopeptide sequence that can bind an MHC molecule encodedby at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more different DRalleles, wherein the oligopeptide sequence comprises AX₁FVAAX₂TLHAAA(SEQ ID NO:2), wherein X₁ is selected from the group consisting of Y, H,I, E, N, Q and K; and X₂ is selected from the group consisting of F, N,Y and W. According to the inventions, the oligopeptide sequence does notcomprise AKFVAAWTLKAAA (SEQ ID NO:3).

In some embodiments, the polypeptides of the present invention compriseone or more copies of one or more amino acid sequences constituting the13 amino acid C-terminal fragment of an oligopeptide selected from SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72.

The pan-DR binding peptides of the present invention are capable ofbinding multiple different MHC alleles and can also be referred to as“helper peptides.” In some embodiments, the pan-DR binding peptides arecapable of binding MHC molecules encoded by 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 or more different MHC class II alleles. In some embodiments theresponse induced is an enhanced immune response. In some embodimentsimmune response is directed towards the pan-DR binding oligopeptides ofthe invention. Alternatively, or in addition, the binding of the pan-DRbinding oligopeptides to an MHC molecule further enhances an immuneresponse to a second antigen, e.g., a polypeptide comprising one or moreCTL peptides and/or a humoral (i.e., antibody) response to a protein ornon-protein antigen.

The pan-DR polypeptide sequences described herein can be used alone, oras part of a larger fusion protein or conjugate. Thus, the polypeptidesof the invention can include additional amino acids at either or bothtermini in addition to the pan-DR peptide sequences set forth herein.For example, it can be desirable to include other amino acid sequencesas tags or markers or to promote in vivo or in vitro stability orotherwise to include additional benefits as is generally understood inthe art. Moreover, the polypeptides of the invention can comprise one,two, three or more copies of one or more pan-DR oligopeptide sequence inthe polypeptide and/or include multiple different pan-DR oligopeptidesequences in the polypeptides of the invention. Moreover, thepolypeptides can include at least one additional HTL oligopeptide and/orCTL oligopeptide and/or antibody-inducing polypeptide, for example whereit is desirable to enhance the immune response to such sequences or topolypeptides comprising such sequences. In such embodiments, the pan-DRsequences described herein function as alternatives to traditionalcarrier proteins such as Keyhole Limpet hemocyanin, tetanus toxoid, anddiphtheria toxoid or as alternatives for prior art universal T-helperepitopes.

The polypeptides of the invention can be of any length. In someembodiments, the polypeptides comprising at least one pan-DR bindingoligopeptide sequence of the invention, and optionally other amino acidsequences, are no longer than e.g., 1000, 900, 800, 700, 600, 500, 400,300, 200, 100, 80, 60, 50, 40, 30, 20, or 15 amino acids, but in manycases the polypeptides comprising the pan-DR binding oligopeptide(s) areof approximately the same length as a native polypeptide, which has beenmodified by introduction of one or a few of the pan-DR bindingoligopeptides of the invention (so that the polypeptide includes amajority of a native polypeptide sequence). Some polypeptides of theinvention including pan-DR binding oligopeptides of the invention are,on the other hand, “multi-epitope constructs”, i.e. polypeptidescomprising a plurality of epitopes derived from one or more antigens,where the epitopes are organized in a non-naturally occurring order witha view to optimizing antigenicity or immunogenicity of the construct.Hence, the multi-epitope expression products discussed below under thedisclosure of the polynucleotides of the invention are also embodimentsof the peptides of the invention.

In such multi-epitope constructs, the plurality of epitopes is typicallyselected from a plurality of CTL epitopes, a plurality of B-cellepitopes, a plurality of T helper lymphocyte epitopes, a plurality ofCTL and B-cell epitopes, a plurality of CTL and T helper lymphocyteepitopes, a plurality of B-cell and T helper lymphocyte epitopes, and aplurality of B-cell, CTL and T helper lymphocyte epitopes. Theseepitopes may be derived from on single antigenic protein, but may alsobe derived from at least 2 different polypeptide antigens, and in turn,these may be from the same or different species (of e.g. bacteria,virus, and parasites).

In some embodiments, the pan-DR binding oligopeptide sequences candiffer from the original sequence by being modified by terminal-NH₂acylation, e.g., by alkanoyl (C₁-C₂₀) or thioglycolyl acetylation,terminal-carboxy amidation, e.g., ammonia, methylamine, etc. In someembodiments, these modifications may provide sites for linking to asupport or other molecule.

The biological activity of the peptides identified above may be assayedin a variety of systems. Typically, the ability to inhibitantigen-specific T cell activation is tested. In one exemplary protocol,an excess of peptide is incubated with an antigen-presenting cell ofknown MHC expression, (e.g., DR1) and a T cell clone of known antigenspecificity (e.g., tetanus toxin 830-843) and MHC restriction (again,DR1), and the immunogenic peptide itself (i.e., tetanus toxin 830-843).The assay culture is incubated for a sufficient time for T cellproliferation, such as four days, and proliferation is then measuredusing standard procedures, such as pulsing with [³H]-thymidine duringthe last 18 hours of incubation. The percent inhibition, compared to thecontrols which do not receive peptide, is then calculated.

The capacity of peptides to inhibit antigen presentation in an in vitroassay has been correlated to the capacity of the peptide to inhibit animmune response in vivo. In vivo activity may be determined in animalmodels, for example, by administering an immunogen known to berestricted to the particular MHC molecule recognized by the peptide, andthe immunomodulatory peptide. T lymphocytes are subsequently removedfrom the animal and cultured with a dose range of immunogen. Inhibitionof stimulation is measured by conventional means, e.g., pulsing with[³H]-thymidine, and comparing to appropriate controls. Certainexperimental details will of course be apparent to the skilled artisan.See also, Adorini, et al., Nature 334: 623-625 (1988), incorporatedherein by reference.

A large number of cells with defined MHC molecules, particularly MHCClass II molecules, are known and readily available from, for instance,the American Type Culture Collection (see, e.g., “Catalogue of CellLines and Hybridomas,” 6th edition (1988) 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A.

Some embodiments of the oligopeptides of the present invention comprisemodifications to the N- and C-terminal residues. As will be wellunderstood by the artisan, the N- and C-termini may be modified to alterphysical or chemical properties of the peptide, such as, for example, toaffect binding, stability, bioavailability, ease of linking, and thelike.

Optionally, modifications of peptides with various amino acid mimeticsor D-amino acids, for instance at the N- or C-termini, can be used, andare useful for instance, in increasing the stability of the peptides invivo. Such peptides may be synthesized as “inverso” or “retroinverso”forms, that is, by replacing L-amino acids of a sequence with D-aminoacids, or by reversing the sequence of the amino acids and replacing theL-amino acids with D-amino acids. As the D-peptides are substantiallymore resistant to peptidases, and therefore are more stable in serum andtissues compared to their L-peptide counterparts, the stability ofD-peptides under physiological conditions may more than compensate for adifference in affinity compared to the corresponding L-peptide. Further,L-amino acid containing peptides with or without substitutions can becapped with a D-amino acid to inhibit exopeptidase destruction of theimmunogenic peptide.

In vivo stability of polypeptides can be assayed in a number of ways.For instance, peptidases and various biological media, such as humanplasma and serum, have been used to test stability. See, e.g., Verhoef,et al., Eur. J. Drug Metab. Pharmacokin. 11:291-302 (1986); Walter, etal., Proc. Soc. Exp. Biol. Med. 148:98-103 (1975); Witter, et al.,Neuroendocrinology 30:377-381 (1980); Verhoef, et al., J. Endocrinology110:557-562 (1986); Handa, et al., Eur. J. Pharmacol. 70:531-540 (1981);Bizzozero, et al., Eur. J. Biochem. 122:251-258 (1982); Chang, Eur. J.Biochem. 151:217-224 (1985), all of which are incorporated herein byreference.

The peptides or analogs of the invention can be modified by altering theorder or composition of certain residues, it being readily appreciatedthat certain amino acid residues essential for biological activity,e.g., those at critical contact sites, may generally not be alteredwithout an adverse effect on biological activity. The non-critical aminoacids need not be limited to those naturally occurring in proteins, suchas L-α-amino acids, or their D-isomers, but may include non-proteinamino acids as well, such as β-γ-δ-amino acids, as well as manyderivatives of L-α-amino acids. An oligopeptide of the present inventioncan comprise either L-amino acids or D-amino acids, but usually notD-amino acids within a core binding region.

The peptides of the invention can be prepared in a wide variety of ways.In some embodiments, recombinant DNA technology is employed wherein anucleotide sequence which encodes an immunogenic polypeptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression. These procedures are generally knownin the art, as described generally in Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989). Alternatively, depending on their size, thepolypeptides can be synthesized in solution or on a solid support inaccordance with conventional techniques. Various automatic synthesizersare commercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young, Solid Phase PeptideSynthesis, 2d. Ed., Pierce Chemical Co. (1984), supra.

III. Polynucleotides

The present invention provides for polynucleotides encoding thepolypeptides of the invention as described herein. For example,polynucleotides encoding a polypeptide comprising an oligopeptidesequence that can bind an MHC molecule encoded by at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or more different DR alleles, wherein theoligopeptide sequence comprises AX₁FVAAX₂TLX₃AA₄A (SEQ ID NO:1), whereinX₁ is selected from the group consisting of W, F, Y, H, D, E, N, Q, Iand K; X₂ is selected from the group consisting of F, N, Y and W; X₃ isselected from the group consisting of H and K, and A₄ is selected fromA, D, and E. In some embodiments, the polypeptide comprises anoligopeptide sequence that can bind an MHC molecule encoded by at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more different DR alleles,wherein the oligopeptide sequence comprises AX₁FVAAX₂TLHAAA (SEQ IDNO:2), wherein X₁ is selected from the group consisting of Y, H, I, E,N, Q and K; and X₂ is selected from the group consisting of F, N, Y andW. According to the invention, the oligopeptide sequence does notcomprise AKFVAAWTLKAAA (SEQ ID NO: 3). In some embodiments, thepolynucleotides of the present invention encode one or more amino acidsequences, each constituting the 13 amino acid C-terminal fragment of aoligopeptide selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, orSEQ ID NO: 72; the polynucleotides may encode polypeptides which includethe sequences or truncates in fusion with other amino acid sequences.So, polynucleotides of the invention are provided that encodepolypeptides, as disclosed herein, comprising two or more CTL and/or HTLoligopeptide sequences, including at least one pan-DR oligopeptidesequence of the invention.

Polynucleotide constructs of the present invention can encode any of theabove-described peptides or polypeptides. For example, multiple class Iand/or class II MHC epitopes and/or B-cell epitopes, for example, morethan one class II MHC epitopes (i.e., at least one pan-DR-bindingoligopeptide sequence as described herein, e.g., SEQ ID NOs: 1, 2 and4-72) or a combination of class I WIC epitopes and class II MHC epitopesand B-cell epitopes. Class I MHC-encoding epitope nucleic acids are alsoreferred to as “CTL epitope nucleic acids,” and class II MHC-encodingepitope nucleic acids are referred to as “HTL epitope nucleic acids”.“B-cell epitopes” are epitopes, either linear or conformational, whichbind antibodies or B-cell receptors. Some multi-epitope constructs canhave a portion of their sequence encoding class I WIC epitopes and/orB-cell epitopes and another portion encoding class II MHC epitopes. Insome embodiments, the CTL epitope nucleic acids encode an epitopepeptide of about eight to about thirteen amino acids in length, e.g.,about eight to about eleven amino acids in length, e.g., about nineamino acids in length. The HTL epitope nucleic acids encode at least onepan-DR oligopeptide as described herein, but where more than one HTLepitope is included, other HTL epitopes can be used as well. In someembodiments, the polynucleotide constructs include, for example, five ormore, ten or more, fifteen or more, twenty or more, or twenty-five ormore epitope nucleic acids. All of the CTL epitope nucleic acids in amulti-epitope polynucleotide construct can be from one organism (e.g.,the nucleotide sequence of every epitope nucleic acid may be present inHIV strains), or the multi-epitope construct can include epitope nucleicacids present in two or more different organisms (e.g., some epitopesfrom HIV and some from HCV). As described hereafter, one or more epitopenucleic acids in the multi-epitope construct may be flanked by a spacernucleic acid.

As will be understood, nucleic acid constructs encoding B-cell epitopecontaining polypeptides linked to or otherwise including the PADREanalogues of the invention are an embodiment of the invention. Suchnucleic acid constructs typically encode a fusion construct where thePADRE analogues present in the expression product do not interferenegatively with the conformation of B-cell epitopes from thepolypeptide. In cases where the B-cell epitopes are linear, the B-cellepitopes may simply be fused to the PADRE analogues, so the provision ofthe encoding nucleic acid is relatively uncomplicated. When the B-cellepitopes are conformational, however, typical examples of introductionpoints for the PADRE analogue encoding nucleic acids are in flexibleloops of the polypeptide or in flexible termini (where 3D-structure ofthe conformational epitopes is sought preserved, e.g. by using entireprotein domains or even entire polypeptides), but it is also possible tointroduce the PADRE analogue encoding nucleic acid in regions encodinge.g. intracellularly confined parts of the polypeptide in question,since these regions are not relevant for immune responses in vivo. Theexpression products of such nucleic acid constructs are useful asantibody inducing immunogens, because the PADRE analogue sequencesintroduced will provide for increased T-lymphocyte help in theelicitation of an immune response against the expression product. Assuch, the expression products may be used directly as immunogens (forantibody induction in e.g. a vaccine or an immunogenic composition usedfor antibody production in animals) or it may exert its effect afterbeing expressed in vivo in an animal which has been subjected to nucleicacid immunization with the encoding nucleic acid.

A “spacer” refers to a sequence that is inserted between two epitopes ina multi-epitope construct to prevent the occurrence of junctionalepitopes and/or to increase the efficiency of processing. Amulti-epitope construct may have one or more spacer nucleic acids. Aspacer nucleic acid may flank each epitope nucleic acid in a construct,or the spacer nucleic acid to epitope nucleic acid ratio may be about 2to 10, about 5 to 10, about 6 to 10, about 7 to 10, about 8 to 10, orabout 9 to 10, where a ratio of about 8 to 10 has been determined toyield favorable results for some constructs.

The spacer nucleic acid may encode one or more amino acids. In someembodiments a spacer nucleic acid flanking a class I WIC epitope in amulti-epitope construct is between one and about eight amino acids inlength, between two and eight amino acids in length, between three andeight amino acids in length, between four and eight amino acids inlength, between five and eight amino acids in length, between six andeight amino acids in length, or between seven and eight amino acids inlength. A spacer nucleic acid flanking a class II MHC epitope in amulti-epitope construct is in some embodiments greater than five, six,seven, or more amino acids in length, and in some embodiments greaterthan five or six amino acids in length.

The number of spacers in a construct, the number of amino acids encodedin a spacer polynucleotide, and the amino acid composition of a spacercan be selected to optimize epitope processing and/or minimizejunctional epitopes. In some embodiments spacers are selected byconcomitantly optimizing epitope processing and junctional motifs.Suitable amino acids for optimizing epitope processing are describedherein. Also, suitable amino acid spacing for minimizing the number ofjunctional epitopes in a construct are described herein for class I andclass II HLAs. For example, spacers flanking class II MHC epitopes canin some embodiments include G, P, and/or N residues as these are notgenerally known to be primary anchor residues (see, e.g.,PCT/US00/19774). In some embodiments a spacer for flanking a class IIMHC epitope includes alternating G and P residues, for example, (GP)n(SEQ ID NOs: 87-95), (PG)n (SEQ ID NOs: 96-104), (GP)nG (SEQ ID NOs: 85,105-112), (PG)nP (SEQ ID NOs: 86, 113-120), and so forth, where n is aninteger between one and ten, between two or about two, and in someembodiments a specific example of such a spacer is GPGPG (SEQ ID NO: 85)or PGPGP (SEQ ID NO: 86). In some embodiment for class I MHC epitopes,the spacer comprises one, two, three or more consecutive alanine (A)residues, optionally preceded by K, N or G.

In some multi-epitope constructs, it is sufficient that each spacernucleic acid encode the same amino acid sequence. In multi-epitopeconstructs having two spacer nucleic acids encoding the same amino acidsequence, the spacer nucleic acids encoding those spacers may have thesame or different nucleotide sequences, where different nucleotidesequences may decrease the likelihood of unintended recombination eventswhen the multi-epitope construct is inserted into cells.

In other multi-epitope constructs, one or more of the spacer nucleicacids may encode different amino acid sequences. While many of thespacer nucleic acids may encode the same amino acid sequence in amulti-epitope construct, one, two, three, four, five or more spacernucleic acids may encode different amino acid sequences, and it ispossible that all of the spacer nucleic acids in a multi-epitopeconstruct encode different amino acid sequences. Spacer nucleic acidsmay be optimized with respect to the epitope nucleic acids they flank bydetermining whether a spacer sequence will maximize epitope processingand/or minimize junctional epitopes, as described herein.

Multi-epitope constructs may be distinguished from one another accordingto whether the spacers in one construct optimize epitope processing orminimize junctional epitopes over another construct, and in someembodiments, constructs may be distinguished where one construct isconcomitantly optimized for epitope processing and junctional epitopesover the other. Computer assisted methods and in vitro and in vivolaboratory methods for determining whether a construct is optimized forepitope processing and junctional motifs are described herein.

In some embodiments, polynucleotide constructs of the invention areprovided as an expression vector comprising a nucleic acid encoding atleast one pan-DR oligopeptide sequence of the invention, and optionallyany or all of the sequences described herein in the context ofpolypeptides. Construction of such expression vectors is described, forexample in PCT/US99/10646, the disclosure of which is hereinincorporated by reference. The expression vectors contain at least onepromoter element that is capable of expressing a transcription unitencoding the nucleic acid in the appropriate cells of an organism sothat the antigen is expressed and targeted to the appropriate MHCmolecule. For example, for administration to a human, a promoter elementthat functions in a human cell is incorporated into the expressionvector.

Basic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994); Oligonucleotide Synthesis. A Practical Approach (Gait, ed.,1984); Kuijpers, Nucleic Acids Research 18(17):5197 (1994); Dueholm, J.Org. Chem. 59:5767-5773 (1994); Methods in Molecular Biology, volume 20(Agrawal, ed.); and Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, e.g., Part I,chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” (1993)).

The nucleic acids encoding the relevant oligopeptide sequences (e.g.,epitopes) can be assembled in a construct according to standardtechniques. In some embodiments, the nucleic acid sequences encodingpan-DR binding oligopeptides, and optionally multi-epitope polypeptides,are isolated using amplification techniques with oligonucleotideprimers, or are chemically synthesized. Recombinant cloning techniquescan also be used when appropriate. Oligonucleotide sequences areselected which either amplify (when using PCR to assemble the construct)or encode (when using synthetic oligonucleotides to assemble theconstruct) the desired epitopes.

Amplification techniques using primers are typically used to amplify andisolate sequences encoding the epitopes of choice from DNA or RNA (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Methods such aspolymerase chain reaction (PCR) and ligase chain reaction (LCR) can beused to amplify epitope nucleic acid sequences directly from mRNA, fromcDNA, from genomic libraries or cDNA libraries. Restriction endonucleasesites can be incorporated into the primers. Multi-epitope constructsamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

Synthetic oligonucleotides can also be used to construct thepolynucleotides of the invention. In some embodiments, this method isperformed using a series of overlapping oligonucleotides, representingboth the sense and non-sense strands of the gene. These DNA fragmentsare then annealed, ligated and cloned. Oligonucleotides that are notcommercially available can be chemically synthesized according to thesolid phase phosphoramidite triester method first described by Beaucage& Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.,12:6159-6168 (1984). Purification of oligonucleotides is by eithernative acrylamide gel electrophoresis or by anion-exchange HPLC asdescribed in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

In some embodiments, the epitopes of polynucleotide constructs of theinvention are subcloned into an expression vector that contains a strongpromoter to direct transcription, as well as other regulatory sequencessuch as enhancers and polyadenylation sites. Suitable promoters are wellknown in the art and described, e.g., in Sambrook et al. and Ausubel etal. Eukaryotic expression systems for mammalian cells are well known inthe art and are commercially available. Such promoter elements include,for example, cytomegalovirus (CMV), Rous sarcoma virus LTR and SV40.

The expression vector typically contains a transcription unit orexpression cassette that contains all the additional elements requiredfor the expression of the polynucleotide construct in host cells. Forexample, an expression cassette can contain a promoter operably linkedto a multi-epitope construct and signals required for efficientpolyadenylation of the transcript. Additional elements of the cassettemay include enhancers and introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

Selectable markers can be incorporated into the expression vectors usedto express the peptides of the invention. These genes can encode a geneproduct, such as a protein, necessary for the survival or growth oftransformed host cells grown in a selective culture medium. Host cellsnot transformed with the vector containing the selection gene will notsurvive in the culture medium. Typical selection genes encode proteinsthat confer resistance to antibiotics or other toxins, such asampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline.Alternatively, selectable markers may encode proteins that complementauxotrophic deficiencies or supply critical nutrients not available fromcomplex media. In some embodiments, the vector will have one selectablemarker that is functional in, e.g., E. coli, or other cells in which thevector is replicated prior to being introduced into the host cell. Anumber of selectable markers are known to those of skill in the art.

A variety of expression vectors can be used to transport the geneticinformation into the cell. Any of the conventional vectors used forexpression in eukaryotic cells may be used. Expression vectorscontaining regulatory elements from eukaryotic viruses are typicallyused in eukaryotic expression vectors, e.g., SV40 vectors, CMV vectors,papilloma virus vectors, and vectors derived from Epstein Bar virus.

The polynucleotide constructs of the invention can be expressed from avariety of vectors including plasmid vectors as well as viral orbacterial vectors. Examples of viral expression vectors includeattenuated viral hosts, such as vaccinia or fowlpox. As an example ofthis approach, vaccinia virus is used as a vector to express nucleotidesequences that encode the (poly)peptides of the invention. Uponintroduction into a host bearing a tumor, the recombinant vaccinia virusexpresses the immunogenic peptide, and thereby elicits a host CTL and/orHTL response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848.

The polynucleotides of the invention can be expressed in a variety ofhost cells, including E. coli, other bacterial hosts, yeast and insectcells, so as to obtain the protein expression products described herein.The host cells can be microorganisms, such as, for example, yeast cells,bacterial cells, or filamentous fungal cells. Examples of suitable hostcells include, for example, Pseudomonas sp., Escherichia sp. (e.g., E.coli), Bacillus, among many others. Suitable yeast cells can be of anyof several genera, including, for example, Saccharomyces (e.g., S.cerevisiae), and Candida. Suitable fungal cells are selected from Pichiaspp., Aspergillus spp, and other fungi suitable for recombinantproduction. Suitable insect cells can be of several varieties,including, for example, S2, Sf9 and Hi-5 cells.

A wide variety of other vectors useful for therapeutic administration orimmunization, e.g. adeno and adeno-associated virus vectors, retroviralvectors, non-viral vectors such as BCG (Bacille Calmette Guerin),Salmonella typhi vectors, detoxified anthrax toxin vectors, and thelike, will be apparent to those skilled in the art. So, the inventionalso relates to prophylactic or therapeutic use of such vectors by meansof administration to an individual in need thereof of such vectors.

Immunogenicity and antigenicity of the multi-epitope constructs areevaluated as described herein.

As set forth above, the present invention also relates to isolatedpolypeptides encoded by any of the polynucleotides of the invention asdescribed herein—as such, these polypeptides are also “peptides” or“polypeptides” of the present invention.

IV. Further Antigens

As explained above, the pan-DR oligopeptides of the invention can beused to enhance a cellular or humoral immune response to a variety ofantigens. Antigenic determinants from such an antigen, or the antigen assuch, can be administered admixed with or linked to the pan-DR bindingpeptides of the present invention. For example, the antigenicdeterminant can be linked or mixed, or administered in series, with thehelper peptides of the present invention to elicit or enhance an immuneresponse. Essentially any antigen can be used (e.g., polysaccharides,proteins, glycoproteins, lipids, glycolipids, lipopolysaccharides andthe like) in combination with the pan-DR binding oligopeptide sequencesof the present invention. Protein antigens may be derived frominfectious agents such as bacteria, fungi, virus, protozoans, helminthsand other parasites, but they may also be disease-associated antigens,such as cancer antigens or antigens which are overexpressed or otherwiseinexpedient in certain disease states. The cancer antigens listed in WO00/20027 are all relevant protein antigens, and further antigens arethose associated with inflammation (here TNF is a good example). Thepresent invention discloses a number of TNF constructs which are allpart of the invention—generally, all these constructs are also part ofthe invention, as are nucleic acid fragments encoding them, vectorscomprising the nucleic acid fragments and host cells including thenucleic acid fragments or vectors (or being transformed with thevectors).

In some embodiments the oligopeptides of the present invention areadministered alone. In some embodiments the oligopeptides of theinvention are administered in conjunction with a second antigenicdeterminant. In some embodiments the oligopeptides of the invention areadmixed with the antigenic determinant. In some embodiments the peptidesof the invention are linked to the antigenic determinant. For example,protein antigens can be linked directly or indirectly to the pan-DRbinding oligopeptides as fusion proteins (via peptide bonding).Antigens, including non-protein antigens, can be linked to the pan-DRoligopeptides, or polypeptides comprising the oligopeptide sequences,via other covalent conjugation methods.

In some embodiments the antigenic determinant administered with thepeptides of the present invention is a protein. In some embodiments theantigenic determinant administered with the peptides of the presentinvention is a polysaccharide. In some embodiments the antigenicdeterminant administered with the peptides of the present invention is aglycoprotein. In some embodiments the antigenic determinant administeredwith the peptides of the present invention is a lipid. In someembodiments the antigenic determinant administered with the peptides ofthe present invention is a glycolipid. In some embodiments the antigenicdeterminant administered with the peptides of the present invention is alipopolysaccharide.

Carbohydrate epitopes include a carbohydrate structure, but can bepresent as a glycoconjugate, e.g., glycoprotein, glycopeptide,glycolipid, and the like, DNA, RNA, or a polysaccharide,oligosaccharide, or monosaccharide against which an immune response isdesired. The carbohydrate epitope may induce a wide range of immuneresponses. One of skill will recognize that various carbohydratestructures exemplified herein can be variously modified according tostandard methods, without adversely affecting antigenicity. Forinstance, the monosaccharide units of the saccharide may be variouslysubstituted or even replaced with small organic molecules, which serveas mimetics for the monosaccharide.

Examples of suitable antigens include those derived from bacterialsurface polysaccharides which can be used in carbohydrate-basedvaccines. Bacteria typically express carbohydrates on their cell surfaceas part of glycoproteins, glycoplipids, 0-specific side chains oflipopolysaccharides, capsular polysaccharides and the like. Exemplarybacterial strains include Streptococcus pneumonia (see, e.g.,WO/2005/120563 and carbohydrate epitopes therein), Neisseriameningitidis, Haemophilus influenza, Klebsiella spp., Pseudomonas spp.,Salmonella spp., Shigella spp., and Group B streptococci.

A number of suitable bacterial carbohydrate epitopes are described inthe prior art (e.g., Sanders, et al. Pediatr. Res. 37:812-819 (1995);Bartoloni, et al. Vaccine 13:463-470 (1995); Pirofski, et al., Infect.Immun. 63:2906-2911 (1995) and International Publication No. WO93/21948) and as described in, e.g., U.S. Pat. No. 6,413,935.

In general, the HLA binding epitopes disclosed in any one of thefollowing international patent applications may according to theinvention be useful and the contents of which are therefore incorporatedby reference herein: WO 93/03764, WO 95/22317, WO 94/03205, WO 95/19783,WO 97/34617, WO 02/20053, WO 94/20127, WO 97/34621, WO 02/20616, WO95/07707, WO 95/04817, WO 98/33888, WO 94/26774, WO 96/03140, WO02/20035, WO 96/40213, WO 97/26784, WO 97/33602, WO 98/32456, WO99/61916, WO 99/45954, WO 99/65522, WO 01/62776, WO 01/00225, WO2004/031211, WO 99/58658, WO 2005/012502, WO 00/44775, WO 02/019986, WO2004/031210, WO 01/21189, WO 01/24810, WO 2005/033265, WO01/42270,WO01/41788, WO01/42267, WO 01/45728, WO 0141787, WO 02/061435, WO02/061435, WO 01/41741, WO 2004/052917, WO 01/36452, WO 03/087126, WO01/47541, WO 02/083714, WO 01/41799, WO 2005/089164, WO 2003/040165, WO2005/120563, WO 2004/094454, WO 2004/053086, WO 2004/089973, WO2008/054540, WO 2008/039267, WO 99/19478, WO 92/21033, WO 94/11738, WO93/22338, WO 95/25530, WO 95/25739, WO 2005/118626, WO 93/03753, WO94/19011, WO 95/03777, WO 95/19783, WO 95/04542, WO 01/42270, WO01/41788, WO 01/42267, WO 01/45728, WO 01/41787, WO 01/41741, WO2004/052917, WO2004/094454, and WO 2004/089973.

V. Preparation of Conjugates

The pan-DR binding peptides of the invention can be linked to at leastone further antigenic determinant in a variety of ways. Ionicinteractions are possible through the termini or through the ε-aminogroup of lysine. Hydrogen bonding between the side groups of theresidues and the antigenic determinants are also possible. In otherembodiments, conformation interactions between the pan-DR bindingpeptide and the antigenic determinant may give rise to a stableattachment.

As noted above, antigenic determinants may be covalently linked to thepan-DR binding peptides to prepare conjugates of the invention. In someembodiments antigenic determinant/pan-DR binding peptide conjugates arelinked by a spacer molecule or linker. In some embodiments, theantigenic determinant may be attached to the pan-DR binding peptidewithout a linker.

The spacer or linker is typically comprised of neutral molecules, suchas, aliphatic carbon chains, amino acids or amino acid mimetics, whichare substantially uncharged under physiological conditions and may havelinear or branched side chains. A number of compositions and methods forlinking various biomolecules are known to those of skill in the art. Anumber of methods for covalently linking a pan-DR binding peptide, forinstance, to a carbohydrate epitope are possible. Methods suitable forlinking pan-DR binding peptides to carbohydrate antigens are disclosedfor instance in WO 93/21948.

A number of linkers are well known and are either commercially availableor are described in the scientific literature. The linking moleculesused in the present invention are of sufficient length to permit the twoportions of the molecule to interact independently and freely withmolecules exposed to them. In the case of carbohydrate epitopes, thelinking molecules are typically 1-50 atoms long. In some embodiments,the linking molecules will be aryl acetylene, ethylene glycol oligomerscontaining 2-14 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other suitable linkers include lipid moleculessuch as ceramide and amino acid residues to which a differentcarbohydrate moiety is linked through the amino acid side chain.

The particular linking molecule used may be selected based upon itschemical/physical properties. The linking molecule has an appropriatefunctional group at each end, one group appropriate for attachment tothe reactive sites on the carbohydrate portion and the other groupappropriate for attachment to the amino acid/peptide portion. Forexample, groups appropriate for attachment to the carbohydrate portionare carboxylic acid, ester, isocyanate, alkyl halide, acyl halide andisothiocyanate. Similar groups would be useful for attachment to theamino acid portion. Appropriate selection of the functional group willdepend on the nature of the reactive portion of the amino acid orpeptide.

In one group of embodiments, alkyl or alkylene groups will be useful aslinking groups and will have 1 to 20 carbon atoms, in some embodimentscontain between 3 to 6 carbon atoms. For instance, linkers comprisingpolyethylene glycol and related structures can be used. The term“polyethylene glycol” is used to refer to those molecules which haverepeating units of ethylene glycol, for example, hexaethylene glycol(HO—(CH₂CH₂O)₅—CH₂CH₂OH). When the term “polyethylene glycol” is used torefer to linking groups, it would be understood by one of skill in theart that other polyethers or polyols could be used as well (i.e,polypropylene glycol or mixtures of ethylene and propylene glycols).

In another group of embodiments, the alkyl or alkylene linking groupswill be perfluorinated, rendering them less susceptible to biologicaldegradation. see, U.S. Pat. No. 5,055,562. In some embodiments linkinggroups will include aminocaproic acid, 4-hydroxy butyric acid,4-mercapto butyric acid, 3-amino-1-propanol, ethanolamine,perfluoroethanolamine, and perfluorohydroxybutyric acid. In someembodiments, the two portions are linked via a polyethylene glycolmoiety.

In some embodiments, the linkers between pan-DR binding peptides andother peptides (for example but not limited to, a pan-DR binding peptideand a CTL or B-cell epitope) can be selected from Ala, Gly, or otherneutral spacers of nonpolar amino acids or neutral polar amino acids. Insome embodiments herein the neutral spacer is Ala. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. In someembodiments, exemplary spacers are homo-oligomers of Ala. When present,the spacer will usually be at least one or two residues, more usuallythree to six residues. In some embodiments the pan-DR binding peptide isconjugated to the CTL or antibody-inducing peptide. In some embodiments,the pan-DR binding peptide is positioned at the amino terminus. Thepeptides can be joined by a neutral linker, such as Ala-Ala-Ala or thelike, and in some embodiments can contain a lipid residue such aspalmitic acid or the like which is attached to alpha and epsilon aminogroups of a Lys residue ((PAM)₂Lys), which is attached to the aminoterminus of the peptide conjugate, typically via Ser-Ser linkage or thelike.

The CTL or antibody-inducing peptide may be linked to the pan-DR bindingpeptide either directly or via a spacer either at the amino or carboxyterminus of the CTL peptide. The amino terminus of either the CTL orantibody inducing peptide or the pan-DR binding peptide can be acylated.In some embodiments, the CTL peptide/pan DR binding peptide conjugatecan be linked to certain alkanoyl (C₁-C₂₀) lipids via one or morelinking residues such as Gly, Gly-Gly, Ser, Ser-Ser as described below.In some embodiments lipid moieties include cholesterol, fatty acids, andthe like.

In some embodiments the pharmaceutical compositions of the invention cancontain at least one component which assists in priming CTL. Lipids havebeen identified as agents capable of assisting the priming CTL in vivoagainst viral antigens. For example but not limited to, steroids such ascholesterol, fatty acids such as palmitic acid residues can be attachedto the sulfhydryl group of a cysteine residue, the alpha and epsilonamino groups of a Lys residue and then linked, e.g., via one or morelinking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to animmunogenic peptide, such as a pan-DR binding peptide. In someembodiments, in place of fatty acids, long chain alkyl groups can belinked through an ether linkage to the final amino acid (e.g., acysteine residue).

The lipidated peptide can be injected, either directly in a micellarform, incorporated into a liposome or emulsified in an adjuvant, e.g.,incomplete Freund's adjuvant. In some embodiments a particularlyeffective immunogen comprises palmitic acid attached to alpha andepsilon amino groups of Lys, which is attached via linkage, e.g.,Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide. See, Deres, et al., Nature 342:561-564(1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primea CTL response to the target antigen. In some embodiments, the inductionof neutralizing antibodies can also be primed with P₃CSS conjugated to apeptide which displays an appropriate epitope and the two compositionscan be combined to more effectively elicit both humoral andcell-mediated responses to infection.

In the case of pan-DR binding peptides conjugated to carbohydrateepitopes, the lipid moieties may be linked to the opposite terminus ofthe peptide (e.g., carbohydrate linked to the C-terminus and lipidlinked to the N-terminus). In some embodiments, both the lipid and thecarbohydrate moieties may be linked to the same end of the peptide. Insome embodiments, the two moieties may be linked to the same linker onthe N-terminus.

VI. Methods of Stimulating an Immune Response

Polypeptide immunogens of the present invention may be expressed,concentrated and purified from expression hosts such as E. coli (orother host cells discussed above) using various methods known to one ofskill in the art—alternatively, the immunogens may be prepared by liquidor solid phase peptide synthesis. Purified polypeptide or peptideimmunogens can be formulated with pharmaceutically-acceptable excipientsfor administration into individuals, for example, to stimulate orenhance an immune response and optionally generate a protective and/ortherapeutic immune response. The phrase “protective and/or therapeuticimmune response” refers to a CTL and/or an HTL and/or antibody responseto a disease related antigen, e.g. derived from an infectious agent,which in some way prevents or at least partially arrests diseasesymptoms, side effects or progression, and clears the infectious agent.In some embodiments, the polypeptides or polynucleotides of theinvention are formulated into vaccines containing an immunologicallyeffective amount of one or more of the peptides of the invention and anappropriate pharmaceutical carrier. Thus, peptides of the invention canbe administered individually or in combination either in a singlecomposition or multiple compositions.

The invention further relates to methods of administering apharmaceutical composition comprising an expression vector of theinvention or a polypeptide derived therefrom to stimulate an immuneresponse. The expression vectors are administered by methods well knownin the art as described in, for example, Donnelly et al. (Ann. Rev.Immunol., 15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859,issued Dec. 3, 1996); Felgner (U.S. Pat. No. 5,703,055, issued Dec. 30,1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21,1997). In one embodiment, the multi-epitope construct is administered asnaked nucleic acid.

A pharmaceutical composition comprising an expression vector of theinvention or a polypeptide derived therefrom can be administered tostimulate an immune response in a subject by various routes including,for example, orally, intravaginally, rectally, or parenterally, such asintravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally, intracisternally or by passive orfacilitated absorption through the skin using, for example, a skin patchor transdermal iontophoresis, respectively. Furthermore, the compositioncan be administered by injection, intubation or topically, the latter ofwhich can be passive, for example, by direct application of an ointmentor powder, or active, for example, using a nasal spray or inhalant. Anexpression vector also can be administered as a topical spray, in whichcase one component of the composition is an appropriate propellant. Thepharmaceutical composition also can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices as described in, forexample, Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis, LiposomeTechnology, Vols. I to III (2nd ed. 1993). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer.

The expression vectors of the invention or a polypeptide derivedtherefrom can be delivered to the interstitial spaces of tissues of ananimal body as described in, for example, Felgner et al., U.S. Pat. Nos.5,580,859 and 5,703,055. Administration of expression vectors of theinvention to muscle is a particularly effective method ofadministration, including intradermal and subcutaneous injections andtransdermal administration. Transdermal administration, such as byiontophoresis, is also an effective method to deliver expression vectorsof the invention to muscle. Epidermal administration of expressionvectors of the invention can also be employed. Epidermal administrationinvolves mechanically or chemically irritating the outermost layer ofepidermis to stimulate an immune response to the irritant (Carson etal., U.S. Pat. No. 5,679,647).

Other effective methods of administering an expression vector of theinvention or a polypeptide derived therefrom to stimulate an immuneresponse include mucosal administration as described in, for example,Carson et al., U.S. Pat. No. 5,679,647. For mucosal administration, themost effective method of administration includes intranasaladministration of an appropriate aerosol containing the expressionvector and a pharmaceutical composition. Suppositories and topicalpreparations are also effective for delivery of expression vectors tomucosal tissues of genital, vaginal and ocular sites. Additionally,expression vectors can be complexed to particles and administered by avaccine gun.

To conclude, the peptides of the present invention are useful forinducing immune responses, both with a view to prophylaxis (i.e. with aview to reducing risk of later disease) and with a view totreatment—however the peptides of the invention are also useful forinducing immune responses experimentally and with a view to inducingantibodies in animals from which antibodies and/or B lymphocytes can beisolated—this in turn allows for later production of monoclonalantibodies and antibody derivatives.

VII. Pharmaceutical Compositions

The polypeptides of the present invention, polynucleotides of theinvention, and pharmaceutical and vaccine compositions thereof, can beadministered to mammals, particularly humans, for prophylactic and/ortherapeutic purposes. The polypeptides of the present invention can beused to elicit and/or enhance immune responses against antigens,including but not limited to, pathogen or cancer-associated orcancer-specific biomolecules (e.g., proteins, carbohydrates, etc.).Examples of diseases which can be treated using the present inventioninclude various bacterial infections, viral infections, fungalinfections, parasitic infections and cancer. As discussed above withrespect to proteinaceous antigens, the cancers associated with thecancer antigens from WO 00/20027 are also examples of diseases as arevarious inflammatory diseases such as rheumatoid arthritis andinflammatory bowel disease.

In some therapeutic applications, the present invention is administeredto an individual already suffering from cancer, inflammatory diseases orinfected with the virus or microorganism of interest. Those in theincubation phase or the acute phase of the disease may be treated withthe present invention separately or in conjunction with othertreatments, as appropriate.

In some therapeutic applications, a composition of the present inventionis administered to a patient in an amount sufficient to elicit aneffective CTL response or humoral response to the microorganism or tumorantigen and to cure, or at least partially arrest, symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend in part on the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician.

Therapeutically effective amounts of the compositions of the presentinvention generally range for the initial immunization that is fortherapeutic or prophylactic administration, from about 1.0 μg to about10,000 μg of polypeptide for a 70 kg patient, e.g., from about 100 toabout 8000 μg, e.g., between about 200 and about 6000 μg. In someembodiments, these doses are followed by boosting dosages of from about1.0 μg to about 3000 μg of peptide pursuant to a boosting regimen overweeks to months depending upon the patient's response and condition bymeasuring specific immune responses.

It should be kept in mind that the compositions of the present inventioncan generally be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, in view of the minimization of extraneous substances and therelative nontoxic nature of the conjugates, it is possible and may befelt desirable by the treating physician to administer substantialexcesses of these compositions.

In some embodiments, the present invention can be used prophylacticallyto prevent and/or ameliorate a particular disease, including but notlimited to, bacterial infections, viral infections, fungal infections,parasitic infections and cancer. Effective amounts are as describedabove. Additionally, one of ordinary skill in the vaccine arts wouldalso know how to adjust or modify prophylactic treatments, asappropriate, for example by boosting and adjusting dosages and dosingregimes.

Formulated vaccines of the invention can be combined with apharmaceutically acceptable adjuvant. The formulated vaccines can be anaqueous solution, a suspension or an emulsion. An immunologicallyeffective amount of each immunogen in the vaccines of the presentinvention is determinable by methods known in the art without undueexperimentation.

The adjuvant can be any pharmaceutically acceptable adjuvant. In someembodiments the adjuvant is an alum based compound. In some embodimentsthe adjuvant is aluminum hydroxide. In others the adjuvant is aluminumphosphate. In some embodiments the adjuvant is EMUNADE®. EMUNADE® is anadjuvant consisting of a combination of oil, water and aluminumhydroxide. In some embodiments the adjuvant is QUIL-A, saponin vaccineadjuvant. In some embodiments the adjuvant is QUIL-A, saponin vaccineadjuvant, plus cholesterol. In some embodiments the adjuvant is anemulsion, such as but not limited to MF59 and PROVAX.

In some embodiments, ISCOM is used as an adjuvant. ISCOM is an acronymfor Immune Stimulating Complex and the technology is described, e.g., inMorein et al. (Nature 308:457-460 (1984)). ISCOMs are lipophilic immunestimulating complexes formed as follows. The polypeptides aresolubilized using standard methods, such as with a non-ionic detergent(e.g., Mega-9, Triton X-100, Octylglucoside, Digitonin, Nonidet P-40,C₁₂E₈, Lubrol, Tween-80). A lipid mixture is added to assist ISCOMformation. The lipid mixture can include a phosphatidyl choline and asynthetic cholesterol. In some embodiments, the mixture is first treatedwith non-ionic detergent at room temperature with stirring, then thelipid mixture (equal parts phosphatidyl choline and cholesterol, forexample) is added and stirring continued. QUIL-A, a saponin vaccineadjuvant (a purified glycoside of saponin) is added to polypeptidecomposition and stirring is continued. Then the non-ionic detergent isremoved (for example, by diafiltration with ammonium acetate). Thematrix of the ISCOM is formed by QUIL-A, a saponin vaccine adjuvant. Themorphology of an ISCOM particle, as viewed by electron microscopy, showsa typical cage like structure of approximately 35 nm in size. The ISCOMformation stage can be refined by the use of tangential flowdiafiltration. ISCOMs present purified antigens in a multimeric formbased on the ability of QUIL-A, a saponin vaccine adjuvant tospontaneously form micelles at a critical concentration and by ahydrophobic/hydophilic link that entrap the purified antigens. Formationof ISCOMs can be verified by electron microscopy to verify that thetypical cage-like structures have been formed. The QUIL-A, a saponinvaccine adjuvant, can be added to give a final concentration of about0.01 to 0.1%. In some embodiments, the final concentration is about0.05%.

The invention also relates to pharmaceutical compositions comprising apharmaceutically acceptable carrier and an expression vector of theinvention or a polypeptide of the invention. Pharmaceutically acceptablecarriers are well known in the art and include aqueous or non-aqueoussolutions, suspensions and emulsions, including physiologically bufferedsaline, alcohol/aqueous solutions or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,lipids or liposomes.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize the expressionvector or increase the absorption of the expression vector. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants suchas ascorbic acid or glutathione, chelating agents, low molecular weightpolypeptides, antimicrobial agents, inert gases or other stabilizers orexcipients. Expression vectors can additionally be complexed with othercomponents such as peptides, polypeptides and carbohydrates. Expressionvectors can also be complexed to particles or beads that can beadministered to an individual, for example, using a vaccine gun. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the expressionvector.

The invention further relates to methods of administering apharmaceutical composition comprising an expression vector of theinvention or a polypeptide derived therefrom to stimulate an immuneresponse. The expression vectors are administered by methods well knownin the art as described in, for example, Donnelly et al. (Ann. Rev.Immunol., 15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859,issued Dec. 3, 1996); Felgner (U.S. Pat. No. 5,703,055, issued Dec. 30,1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21,1997). In one embodiment, the polynucleotides of the invention areadministered as naked nucleic acid.

The compositions of the invention are also useful for induction ofimmune responses, e.g. in experimental animals or in animals with a viewto prepare antibodies or antibody derivatives. In such compositions therequirement that the composition should be pharmaceutically acceptableis of minor importance, and it is e.g. possible to use adjuvants notconsidered suitable for use in humans.

PREAMBLE TO EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Proteolytic cleavage within PADRE (SEQ ID NO: 3) has been observed inPADRE-containing proteins, in particular around the two lysine residuesK2 and K10 but also around W7. Cleavage within PADRE is undesirablebecause it, in recombinantly produced proteins, leads to heterogeneity,reduced production yield and altered stability. Even if cleavage can besuccessfully avoided by the use of additives during upstream anddownstream processes, a resulting PADRE containing vaccine may stillhave poor immunogenicity and pharmacokinetics properties such as reducedhalf-life and improper presentation of the T helper epitope(s). Thusthere is a need to remove protease sensitive amino acids/sequences fromthe PADRE sequence.

Provision of PADRE analogues having reduced susceptibility to proteaseactivity will reduce the susceptibility of recombinant expressionproducts to proteases especially in expression systems such as E. coliand Drosophila expression systems in order to avoid proteolyticcleavages within the pan DR binding peptide amino acid sequence duringupstream and/or downstream processes of protein production. Further, inthe event a PADRE analogue is administered to humans, either in isolatedform or as part of an immunogenic polypeptide, the reducedsusceptibility will entail the further advantage of improvedpharmacokinetic properties (i.a. longer serum half-life) and possiblyimproved immunogenicity.

On this basis a number of analogues of SEQ ID NO: 3 have been preparedand subjected to the following sequence of identification steps in orderto identify improved pan DR binding peptides which are less prone toproteolytic degradation:

1) Preparation of library of synthetic PADRE analogues

2) Assaying and selecting analogues from 1) for good HLA bindingproperties

3) Assaying and selecting analogues selected in 2) for in situ proteaseresistance

4) Assaying and selecting analogues selected in 3) for good immunogenicproperties.

Example 1 Testing for HLA-DR Binding of PADRE Analogues

A total of 69 different PADRE analogues (SEQ ID NOs: 4-72) weresynthesized by standard solid phase peptide synthesis—all these PADREanalogues are constituted by, from the N- to the C-terminus, two alanineresidues followed by residues 3-15, which is an analogue of SEQ ID NO: 3(except for SEQ ID NO: 4, which is PADRE preceded by two alanineresidues). The reason for including the 2 extra N-terminal alanines wasto ensure that the isolated peptides would be able to demonstrateendoprotease resistance in an in vitro assay for protease resistance,whereas the immunogenic properties of the PADRE analogues reside in the13 C-terminal amino acids.

The peptides were tested for binding to HLA-DR according to thefollowing procedure:

Peptide/HLA-DR Binding Assays

The standard operating procedure for the class II MHC binding assay isdescribed in Sidney et al. (1998), Current Protocols in Immunology,18.3.1-18.3.19, 1998. Briefly, purified human class II molecules [5 to500 nM] were incubated with various unlabeled peptide binding inhibitorsand 1-10 nM ¹²⁵I-radiolabeled probe peptides for 48 h in PBS containing5% DMSO in the presence of a protease inhibitor cocktail. Finaldetergent concentration in the incubation mixture was 0.05% NonidetP-40. Assays were performed at pH 7.0 with the exception of DR3, whichwas performed at pH 4.5, and DRw53, which was performed at pH 5.0. ThepH was adjusted as described in Sette et al. (1992), J Immunol,148:844-51.

Instead of using HPLC methodology to measure peptide binding to MHCmolecules as detailed in Sidney et al., an anti-MHC class II antibodycoated plate-based capture assay was utilized. This assay has beendeveloped to use a 96-well white polystyrene microtiter platespecifically designed for high-volume, in-plate, radiometric assays.Measurement of the ¹²⁵I-labeled peptide bound to MHC is accomplishedusing the TOPCOUNT (Perkin-Elmer Instruments) benchtop microplatescintillation and luminescence counter, which allows for a highlysensitive assay for high throughput performance.

Peptide binding inhibitors were tested at concentrations ranging from 30μg/ml to 300 pg/ml. Utilizing commercially available curve-fittingalgorithms, the data were plotted in silico and the dose yielding 50%inhibition (IC₅₀) was determined. In appropriate stoichiometricconditions, the IC₅₀ of an unlabeled test peptide to the purified DRmolecule is a reasonable approximation of the affinity of interaction(K_(D)). Peptides were tested in two to four completely independentexperiments.

Results

The binding affinities as indicated via the IC₅₀ values are listed foreach peptide in FIG. 1. On the basis of these IC₅₀ values, it wasconcluded that a fraction of the analogues (38 PADRE analogues)exhibited sufficiently good HLA binding capabilities (shown in FIG. 1 bybold and underlining). Of these good binders, 10 were initially selectedfor further testing for protease resistance: SEQ ID NOs: 4, 10, 19, 23,29, 38, 46, 52, 67, and 69.

It is also important to note that the inclusion of the two N-terminalalanines in SEQ ID NOs. 4-69 did not affect the HLA binding propertiesas evidenced by the similar binding characteristics exhibited by SEQ IDNOs: 3 and 4.

Example 2 Protease Resistance Testing of Selected PADRE Analogues

In order to test the protease resistance of selected, good binding PADREanalogues, their sequences were inserted by cloning techniques into aloop (loop EF) in human TNFα. Cell-free protein crude extract from E.coli expressing the modified TNFα molecules was prepared and proteolyticcleavage within the PADRE analogue sequences was analysed by SDS-PAGEfollowed by immunoblotting.

Full-length, membrane-bound TNFα is a 233 amino-acid long protein. Theamino-terminal fragment 1-76 is absent in the 157 amino-acid longsoluble form of TNFα (77-233).

The template used for the design of human TNFα vaccine candidates is thesoluble form of TNFα (fragment 77-233, SEQ ID NO: 73), preceded by anN-terminal Met for expression in E. coli. Furthermore a point mutationwas made (Y87S) in order to abolish binding of TNFα to its tworeceptors, thereby abolishing the cytotoxicity of the protein. Thus, thevaccine candidates are based on the 158 amino acid residue long SEQ IDNO: 74:

TNF 37.87 (SEQ ID NO: 75) is a TNF variant where PADRE is insertedbetween A₁₈₅ and E₁₈₆ ( . . . PEGA-EAK . . . ). Since PADRE begins withan A, the A was not duplicated, resulting in an insertion of 12 aminoacids instead of 13:

. . . PEGA-AKFVAAWTLKAAA-EAK . . . → PEG-A-KFVAAWTLKAAAEAK . . .

The TNF variants initially tested for protease resistance are thus SEQID NO: 75-TNF 37.87-reference protein comprising “traditional” PADRE:

MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAKFVAAWTLKAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALas well as 9 PADRE analogue containing TNF variants, each similarlyhaving the 12 C-terminal amino acid residues of SEQ ID NOs: 4, 10, 19,23, 29, 38, 46, 52, 67, and 69, respectively, at the underlinedposition:

TNF 37.87-007 sequence: SEQ ID NO: 76MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAWFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL TNF 37.87-016 sequence: SEQ ID NO: 77MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAFFVAANTLKADAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGHAL TNF 37.87-020 sequence: SEQ ID NO: 78MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAYFVAAFTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL TNF 37.87-026 sequence: SEQ ID NO: 79MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAHFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGHAL TNF 37.87-035 sequence: SEQ ID NO: 80MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAHFVAAFTLKAEAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL TNF 37.87-043 sequence: SEQ ID NO: 81MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAEFVAAWTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL TNF 37.87-049 sequence: SEQ ID NO: 82MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGANFVAAYTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGHAL TNF 37.87-064 sequence: SEQ ID NO: 83MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAQFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGHAL TNF 37.87-066 sequence: SEQ ID NO: 84MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAIFVAAWTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

In all sequences, double underlining denotes the initial methionyl andthe Y87S mutation, respectively, and single underlining denotes the panDR binding sequence inserted; bold indicates amino acid changes in thepan DR binding peptide relative to PADRE (SEQ ID NO: 3).

Cloning, Expression, Purification and Characterization of RecombinantTNFα Variants

A synthetic cDNA encoding TNF (Y87S) was used as template.Oligonucleotides encoding PADRE or PADRE analogues were inserted intothe template between A₁₈₅ and E₁₈₆ by PCR (cf. above). Resulting cDNAswere cloned into pET28b+(Novagen) and transformed into E. coli strainHMS 174. For recombinant expression, the resulting E. coli strains werecultured in fermentors in a defined medium at 37° C. Induction ofrecombinant expression was initiated by the addition of 1 mM of IPTG andtemperature was lowered to 25° C. Cultures were harvested at 4, 8 or 24h post-IPTG induction. TNFα variants were purified to >90% homogeneityby affinity chromatography using a TNFα specific monoclonal antibody asdescribed in Nielsen et al., (2004), J Biol. Chem. 279:33593-33600. Thepurified TNFα variants were characterized by MALDI-TOF mass spectrometryto verify their identity and integrity.

In Situ Protease Assay: Analysis of the Protease Resistance of TNF-αVariants Using Western Blot

Aliquots (1 ml) of E. coli cell cultures, expressing the various TNF-αvariants to be tested, were centrifuged (5000 g/5 min/5° C.) and theculture medium was discarded. Cell pellets were resuspended in 1 ml of20 mM Bis-Tris, pH 6.0. Cell disruption was achieved by sonication (4×15sec at an amplitude of 11 μm with 20 sec pauses between each sonicationround). The resulting suspensions were centrifuged (20000 g/30 min/8°C.) and the supernatants (crude extracts) were transferred to freshtubes. Crude extracts were then analysed for protein content using aBradford protein assay (Biorad). Equivalent amounts of proteins wereloaded onto 4-12% NuPAGE gels (Novex). After SDS-PAGE, proteins weretransferred onto nitrocellulose membrane by semi-dry blotting. Forimmunoreaction, incubation with rabbit TNFα antiserum (1 h; dilution1:10 000) and with horse radish peroxidase-conjugated secondary antibody(Dako P448) were carried out in Tris 50 mM, NaCl 150 mM, EDTA 5 mM,Igepal 0.1%, gelatine 0.5%. Detection was with ECL™ reagents (GEHealthcare).

Results

Of the tested TNF variants, three (SEQ ID NOs: 78, 79 and 84) were shownto be protease resistant in the protease assay (data not shown),evidencing that these TNF variants are not sensitive to proteolyticcleavage inside the PADRE analogue sequence.

It was concluded that PADRE analogues defined by at least the 13C-terminal amino acid residues of each of SEQ ID NOs: 23, 29 and 69exhibit a desirable increased resistance towards proteolytic degradationand that proteins modified by including the 13 C-terminal amino acidresidues of SEQ ID NOs: 23, 29 and 69 will exhibit superior yields whenproduced recombinantly (because no or only limited fragmentation of theexpression product will occur), provide for a homogenous expressionproduct and possibly provide for increased immunogenicity because of theincreased biological half-life of a protease resistant immunogen.

Example 3 Immunogenicity Testing of Selected PADRE Analogues

The three protease resistant PADRE analogues identified in Example 2were tested for their immunogenic properties. In one line ofexperiments, the peptides were tested as free peptides in PBMCs(peripheral blood mononuclear cells) from human donors and in two mousestrains (DR4 and bxd). In the second line of experiments, the PADREanalogues were tested in the context of the protease resistant TNFvariants identified in Example 2.

Test of Peptides in Human PBMCs

Human PBMCs (peripheral blood mononuclear cells) were stimulated withthe peptides with sequences identical to the 13 amino acid residues ofSEQ ID NOs: 23, 29 and 69 (in the following these 3 13-mers are termedPADRE.Y2.F7.H10, PADRE H2.N7.H10 and PADRE.I2.H10, respectively). Atdays 1 and 4 IL-2 is added. At day 7, a portion of the cells were takenfor an IFN-γ ELISPOT assay. The remaining cells were restimulated. Atdays 8 and 11 IL-2 was added. At day 14, CD4 cells were purified andtested in an IFN-γ ELISPOT assay.

The IFN-γ ELISPOT assay was performed essentially as described in TangriS et al. (2005), J Immunol 174, 3187-96.

Test of Peptides in Mice

Groups of 3 individuals of each of the HLA-DR4 transgenic and bxd mousestrains were immunized with 20 μg (6 μl of 20 mg/ml peptide+294 μl PBS1×+300 μl CFA) or 2 μg (6 μl of 2 mg/ml peptide+294 μl PBS 1×+300 μlCFA) peptide in CFA at the base of the tail. 10 to 14 days later thespleens were harvested, CD4 cells purified and tested in an IFN-γELISPOT assay (as described in McKinney et al. (2004), J Immunol 173,1941-50). The peptides tested were identical to those tested in humanPBMCs.

Tests of TNFα Variants in Mice

Groups of three HLA-DR4 transgenic-mice were immunized at the base ofthe tail with 10 μg of TNF-derived antigen/mouse in 100 μl of completeFreund's adjunvant (CFA). The animals were boosted 14 days after thefirst immunization using the same dose of antigen in 100 μl of IFA(incomplete Freund's adjuvant). Sera were collected 14 days after eachimmunization for determination of TNFα specific antibody titers. Animalswere then sacrificed for IFN-γ ELISPOT assays.

Determination of TNFα Specific Antibody Titers in Mice Antisera

TNFα specific antibody titers were determined using a direct ELISA.96-well plates (Maxisorb, Nunc) were coated with recombinant TNFαvariant TNF Y87S (5 μg/ml in a carbonate buffer at pH 9.6, 100 μl/well,overnight at 4° C.). Plates were then washed three times and incubatedfor 2 hrs at 37° C. with 200 of blocking buffer (phosphate buffer salinecontaining 1% of bovine serum albumin and 0.05% of Tween 20). Plateswere washed three times and pooled sera from mice immunized with humanTNFα variants were titrated in six steps, using 1/10-dilution steps(starting dilution is 1/1000) in blocking buffer in a total volume of100 μl/well samples were titrated in duplicates. Serum and control wereincubated for 2 hr at 37° C. Plates were washed three times and 100 μlof a biotinylated goat anti-mouse IgG (diluted 1/10000 in blockingbuffer) was transferred to each well and incubated for 1 hr at 37° C.Finally plates were washed three times and incubated for 45 min at roomtemperature with 100 of avidin-peroxidase complex (Vectastain EliteVector PK-6100). Plates were washed again and developed using TMBS. Thereaction was stopped after 10-20 min with 100 μl of 4N H₂SO₄ and A₄₅₀values were determined using an ELISA reader. Antibody titers weredefined as the antiserum dilution yielding an A₄₅₀ value of 0.5.

Results

The results from the test of the peptides in human PBMCs revealed thatPADRE.Y2.F7.H10 and PADRE.I2.H10 provided for superior immune responsesin the IFN-γ ELISPOT assay at both day 7 and day 14 compared to SEQ IDNO: 3, whereas PADRE.H2.N7.H10 provided for immune responses of the sameorder of magnitude as did SEQ ID NO: 3.

In the tests of PADRE analogue peptides in mice, all 3 tested analoguesperformed better than PADRE at the 20 μg dose in DR4 mice, whereas onlythe PADRE.I2.H10 peptide provided for improved immune responses in DR4transgenic mice at the 2 μg dose as measured by the ELISPOT assay. Inthe bxd mouse model, all 3 tested PADRE analogues provided forsignificant immune responses compared to PADRE in the ELISPOT assay atboth doses, without however providing for an improvement of the immuneresponse compared to PADRE.

Immunizations with TNFα variants including the PADRE analogues providedfor the following results:

The HTL induction in the HLA DR4 transgenic mice as measured by IFN-γELISPOT revealed that all TNFα-variants induced HTLs for the PADREanalogue introduced into TNFα and that PADRE.Y2.F7.H10 and PADRE.I2.H10were superior to PADRE in this respect (where PADRE. H2.N7.H10 providedabout the same HTL induction as PADRE). Further, all the variantsprovided for antibody titers significantly higher than those induced bywild type TNFα—the variants containing PADRE.Y2.F7.H10 and PADRE.I2.H10were superior compared to PADRE, and again PADRE. H2.N7.H10 provided animmune response comparable to PADRE. In fact, when correlating antibodytiters with HTL induction, there was a clear linear correlation.

Example 4 Selection and Test of 8 Further PADRE Analogues

Based on the identification of the pan DR binding, protease resistantand immunogenic PADRE analogues PADRE.Y2.F7.H10, PADRE.H2.N7.H10 andPADRE.I2.H10, it was decided to test further PADRE analogues identifiedas good binders in Example 1. The peptides tested were those having the13 C-terminal amino acid residues from SEQ ID NOs. 15 (PADRE.F2.N7.H10),21 (PADRE.Y2.H10), 24 (PADRE.Y2.N7.H10), 26 (PADRE.H2.H10), 47(PADRE.E2.Y7.H10), 54 (PADRE.N2.N7.H10), 66 (PADRE.Q2.F7.H10), and 72(PADRE.I2.N7.H10).

Three of these sequences were shown to be protease resistant accordingto the procedure set forth in Example 2, namely PADRE.E2.Y7.H10,PADRE.N2.N7.H10 and PADRE.Q2.N7.H10.

CONCLUSIONS

The examples described above have demonstrated the existence of a numberof PADRE analogues with improved characteristics in terms of proteaseresistance and/or immunogenicity. It is demonstrated that carefulmodifications in SEQ ID NO: 3 provides for immunogens which are morestable than PADRE, both when being recombinantly expressed and whenbeing used as in vivo immunogens.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

SEQUENCES

SEQ ID NO: 1: AX₁FVAAX₂TLX₃AX₄A SEQ ID NO: 2: AX₁FVAAX₂TLHAAASEQ ID NO: 3: AKFVAAWTLKAAA SEQ ID NO: 4: AAAKFVAAWTLKAAA SEQ ID NO: 5:AAAWFVAAWTLKAAA SEQ ID NO: 6: AAAKFVAAWTLHAAA SEQ ID NO: 7:AAAWFVAAWTLHAAA SEQ ID NO: 8: AAAWFVAAYTLHAAA SEQ ID NO: 9:AAAWFVAAFTLHAAA SEQ ID NO: 10: AAAWFVAANTLHAAA SEQ ID NO: 11:AAAFFVAAWTLKAAA SEQ ID NO: 12: AAAFFVAAWTLHAAA SEQ ID NO: 13:AAAFFVAAYTLHAAA SEQ ID NO: 14: AAAFFVAAFTLHAAA SEQ ID NO: 15:AAAFFVAANTLHAAA SEQ ID NO: 16: AAAFFVAAWTLKADA SEQ ID NO: 17:AAAFFVAAYTLKADA SEQ ID NO: 18: AAAFFVAAFTLKADA SEQ ID NO: 19:AAAFFVAANTLKADA SEQ ID NO: 20: AAAYFVAAWTLKAAA SEQ ID NO: 21:AAAYFVAAWTLHAAA SEQ ID NO: 22: AAAYFVAAYTLHAAA SEQ ID NO: 23:AAAYFVAAFTLHAAA SEQ ID NO: 24: AAAYFVAANTLHAAA SEQ ID NO: 25:AAAHFVAAWTLKAAA SEQ ID NO: 26: AAAHFVAAWTLHAAA SEQ ID NO: 27:AAAHFVAAYTLHAAA SEQ ID NO: 28: AAAHFVAAFTLHAAA SEQ ID NO: 29:AAAHFVAANTLHAAA SEQ ID NO: 30: AAAKFVAAWTLKADA SEQ ID NO: 31:AAAHFVAAWTLKADA SEQ ID NO: 32: AAAHFVAAYTLKADA SEQ ID NO: 33:AAAHFVAAFTLKADA SEQ ID NO: 34: AAAHFVAANTLKADA SEQ ID NO: 35:AAAKFVAAWTLKAEA SEQ ID NO: 36: AAAHFVAAWTLKAEA SEQ ID NO: 37:AAAHFVAAYTLKAEA SEQ ID NO: 38: AAAHFVAAFTLKAEA SEQ ID NO: 39:AAAHFVAANTLKAEA SEQ ID NO: 40: AAADFVAAWTLKAAA SEQ ID NO: 41:AAADFVAAWTLHAAA SEQ ID NO: 42: AAADFVAAYTLHAAA SEQ ID NO: 43:AAADFVAAFTLHAAA SEQ ID NO: 44: AAADFVAANTLHAAA SEQ ID NO: 45:AAAEFVAAWTLKAAA SEQ ID NO: 46: AAAEFVAAWTLHAAA SEQ ID NO: 47:AAAEFVAAYTLHAAA SEQ ID NO: 48: AAAEFVAAFTLHAAA SEQ ID NO: 49:AAAEFVAANTLHAAA SEQ ID NO: 50: AAANFVAAWTLKAAA SEQ ID NO: 51:AAANFVAAWTLHAAA SEQ ID NO: 52: AAANFVAAYTLHAAA SEQ ID NO: 53:AAANFVAAFTLHAAA SEQ ID NO: 54: AAANFVAANTLHAAA SEQ ID NO: 55:AAANFVAAWTLKADA SEQ ID NO: 56: AAANFVAAYTLKADA SEQ ID NO: 57:AAANFVAAFTLKADA SEQ ID NO: 58: AAANFVAANTLKADA SEQ ID NO: 59:AAANFVAAWTLKAEA SEQ ID NO: 60: AAANFVAAYTLKAEA SEQ ID NO: 61:AAANFVAAFTLKAEA SEQ ID NO: 62: AAANFVAANTLKAEA SEQ ID NO: 63:AAAQFVAAWTLKAAA SEQ ID NO: 64: AAAQFVAAWTLHAAA SEQ ID NO: 65:AAAQFVAAYTLHAAA SEQ ID NO: 66: AAAQFVAAFTLHAAA SEQ ID NO: 67:AAAQFVAANTLHAAA SEQ ID NO: 68: AAAIFVAAWTLKAAA SEQ ID NO: 69:AAAIFVAAWTLHAAA SEQ ID NO: 70: AAAIFVAAYTLHAAA SEQ ID NO: 71:AAAIFVAAFTLHAAA SEQ ID NO: 72: AAAIFVAANTLHAAA SEQ ID NO: 73:VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIALSEQ ID NO: 74: MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPD YLDFAESGQVYFGIIALSEQ ID NO: 75: MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAKFVAAWTLKAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 76:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAWFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 77:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAFFVAANTLKADAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 78:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAYFVAAFTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 79:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAHFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 80:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAHFVAAFTLKAEAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 81:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAEFVAAWTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 82:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGANFVAAYTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 83:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLSAIKSPCQRETPEGAQFVAANTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 84:MVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSSQTKVNLLSAIKSPCQRETPEGAIFVAAWTLHAAAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

What is claimed is:
 1. An isolated oligopeptide that can bind to atleast three different HLA-DR alleles with an IC₅₀ value of less than 100nM, wherein the oligopeptide sequence comprises AX₁FVAAX₂TLX₃AX₄A (SEQID NO: 1), wherein X₁ is H; X2 is selected from the group consisting ofF, N, Y and W; and X₃ is H; and X₄ is selected from the group consistingof A, D and E.
 2. The isolated oligopeptide of claim 1, wherein X₁ is H;X₂ is selected from the group consisting of F, N, Y and W; and X₃ is H;and X₄ is A.
 3. The isolated oligopeptide of claim 1, having the aminoacid sequence identical to the 13 amino acid residue C-terminal fragmentof SEQ ID NO:
 29. 4. An isolated polypeptide comprising an oligopeptidesequence that can bind to at least three different HLA-DR alleles withan IC₅₀ value of less than 100 nM, wherein the oligopeptide sequencecomprises AX₁FVAAX₂TLX₃AX₄A (SEQ ID NO:1), wherein X₁ is H; X₂ isselected from the group consisting of F, N, Y, and W; and X₃ is H; andX₄ is selected from the group consisting of A, D and E.
 5. The isolatedpolypeptide of claim 4, wherein X1 is H; X2 is selected from the groupconsisting of F, N, Y and W; and X3 is H; and X4 is A.
 6. The isolatedpolypeptide of claim 4, wherein the oligopeptide consists of the aminoacid sequence identical to the 13 amino acid residue C-terminal fragmentof the oligopeptide consisting of SEQ ID NO:
 29. 7. The isolatedpolypeptide of claim 4, wherein the polypeptide, in addition to theoligopeptide, includes a majority of a native polypeptide sequence. 8.The isolated polypeptide of claim 7, wherein the native polypeptidesequence is from human TNFα.
 9. A composition comprising a polypeptideaccording to claim 4 and an antigen.
 10. The composition of claim 9,wherein the antigen is a second polypeptide.
 11. A compositioncomprising a physiologically acceptable excipient and a polypeptide asdefined in claim
 4. 12. The composition of claim 11, further comprisingan antigen.